CN101816653A - Application of berberine in preparing tumor radio sensitization medicine - Google Patents
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Abstract
The invention discloses a tumor radio sensitization medicine which has an active component of berberine. Proved by research: the berberine has the following efficacy of: 1, breast cancer cells are treated by berberine, which can induce dose-dependently G0/G1 stage cell retardation and apoptosis; 2, the berberine remarkably reduces the expression level of cell cycle control associated protein cyclin B and apoptosis resisting protein Bcl-2 and increases the expression level of apoptins Bax and caspase-3; and 3, the breast cancer cells treated by the berberine remarkably improve the sensitiveness of the cells to radiotherapy. The tumor radio sensitization medicine can improve the treatment effect of tumor radiotherapy, lower the exposure dose of the radiotherapy of a tumor patient, and lower the toxic and side effects brought by the radiotherapy, thereby improving the living quality of patients.
Description
Technical field
The invention belongs to the tumor radio sensitization medicine field, be specifically related to berberine as the application in the preparation tumor radio sensitization medicine.
Background technology
In the prior art, radiotherapy hypersitization medicine commonly used comprises:
(1) MISO (Misonodaxole): MISO is an anoxic cell sensitizer that obtains affirming fully in the clinical experiment stage.
(2) KANGLAITE: be from the Chinese medicine Semen Coicis, to extract the injection of making.This medicine has been widely used in treating various malignant tumor, also is a kind of radiosensitizer simultaneously.
(3) SR-2508 (Etanidacole): SR-2508 is the amide derivatives of nitroimidazole, and it is close with MISO that its enhanced sensitivity is tired, but toxicity is lower, and maximum can tolerate accumulated dose and be about 3 times of MISO.SR-2508 also has sensitization to chemotherapeutic.
(4) AK-2123:AK-2123 is a nitro glyoxaline compound, is a kind of electrophilicity radiosensitizer, the interior injection of oral or tumor.Its enhancement effect and MISO are close, and toxicity is much lower.In addition, animal experiment study is all pointed out and is used AK-2123 that thermotherapy and chemotherapy are also had sensitization.
(5) 912:912 is a kind of anticarcinogen that extracts from Chinese traditional medicine sleeper (Lumbricus), and isolated experiment and integral experiment all have certain radiosensitizing effect.Clinical test results shows that 912 have no side effect.
(6) Radix Ilicis Pubescentis: Radix Ilicis Pubescentis is a kind of holly plant, has effects such as blood circulation promoting and blood stasis dispelling, heat-clearing and toxic substances removing.Clinical research shows that it has certain radiosensitizing effect to the nasopharyngeal carcinoma patient; The average radiological agent amount of being when lymphonodi cervicales disappears also is starkly lower than simple radiation group; Can improve nasopharyngeal carcinoma patient's survival rate.The Radix Ilicis Pubescentis combined radiotherapy is safe, not only can not increase skin and gastrointestinal reaction, and reaction also has certain effect that alleviates to mucous membrane of oropharynx.
(7) lycium barbarum polysaccharide: lycium barbarum polysaccharide is the main component of extracting from the Chinese medicine Fructus Lycii, to the body free of toxic effects.Lycium barbarum polysaccharide is used does not separately have obvious inhibitory action to tumor, but zoopery and clinical observation show that lycium barbarum polysaccharide and radiotherapy use in conjunction have effect of enhanced sensitivity.Clinical research shows that lycium barbarum polysaccharide merging radiotherapy increases to the short term effect of primary lung cancer.
(8) retrandrine: retrandrine extracts from the Chinese medicine Radix Stephaniae Tetrandrae.The Shanghai first medical science tumour hospital height makes the mountain report, adopting retrandrine to share the little dose of amount of being lonizing radiation observes 97 routine advanced lung cancers, exit dose is that 1500～2000rad (convention amount is 6000rad) lung tumors does not dwindle the person in various degree and accounts for 61.9% as a result, shows that retrandrine has certain radiosensitizing effect.
(9) paclitaxel (paclitaxel, trade name taxol): paclitaxel is the natural product of separating in a kind of bark of Ramulus et folium taxi cuspidatae, is the antitumor drug of a new generation.Studies show that it has obvious curative effects to ovarian cancer, breast carcinoma, nonsmall-cell lung cancer, malignant lymphoma etc.In addition, discovering when paclitaxel and ray share has radiosensitizing effect preferably, and sensitization increases and increases along with drug level.
It is a kind of quaternary ammonium compounds that extracts from cohosh Rhizoma Coptidis, Cortex Phellodendri that berberine has another name called berberine, and molecular weight is 317.8.At the nations of China and India berberine is a kind of very ancient medicine, be used for for a long time clinically clinical analgesic, the detoxifcation and anti-enterobacterial infection.Recent study finds that berberine has other biochemistry and the pharmacological activity that comprises blood pressure lowering, anti-myocardial remodelling, heart failure resistance, anti-angiogenic atherosis, blood fat reducing, blood sugar lowering, mediated cell immunity.Research finds that also heavy dose of berberine has antitumor efficacy, the hypertrophy that can suppress colon cancer cell, suppress the esophageal carcinoma, the growth of breast carcinoma and prostate gland cancer cell, but simultaneously can toxicity during heavy dose of the use, toxicity can be eliminated though reduce dosage, antitumous effect can't be produced when dosage is little.Do not see at present the application report of berberine simultaneously as radiotherapy hypersitization medicine.
Summary of the invention
The object of the invention provides the new purposes of berberine, i.e. the application of berberine in the preparation tumor radio sensitization medicine.
Another object of the present invention provides a kind of tumor radio sensitization medicine.
For achieving the above object, the technical solution used in the present invention is: a kind of tumor radio sensitization medicine, described tumor radio sensitization medicine active component is a berberine.
In the technique scheme, described tumor radio sensitization medicine also comprises and well known to a person skilled in the art auxiliary ingredients, for example, when tumor radio sensitization medicine is made the local injection dosage form, can add the vinyl pyrrolidone hydrosol to play slow releasing pharmaceutical; The dosage form of described tumor radio sensitization medicine comprises: intravenous form, local injection dosage form, tablet, multi-pharmaceuticss such as drop.
To discovering of the described tumor radio sensitization medicine of technique scheme:
(1) berberine processing breast cancer cell can the dependent G of inductive dose
0/ G
1Phase cell retardance and apoptosis: berberine suppresses the dosage (IC of the growth of 50%MCF-7 cell and MDA-MB-231 cell
50) be approximately 10 μ mol/L (48 hours) and about 5 μ mol/L (72 hours).
(2) berberine has obviously reduced the expression of cell cycle control associated protein cyclin B and anti-apoptotic protein B cl-2, and has increased the expression of apoptotic proteins Bax and caspase-3.These provide mechanism of action for berberine as tumor radio sensitization medicine.
(3) breast cancer cell after berberine is handled has obviously improved cell to radiocurable sensitivity: MDA-MB-231 cell and MCF-7 cell radiation sensitivity ratio that berberine concentration 5 μ mol/L handled in 24 hours are respectively 1.4207 and 1.5420.
Above-mentioned result of study shows, berberine can be as the agent of preparation tumor radio sensitization, and when berberine concentration in the tumor radio sensitization agent is low to moderate 5 μ mol/L, and pair cell can toxigenicity; And as well known to those skilled in the art, when berberine concentration was low to moderate 5 μ mol/L, the processing of tumor cell being carried out the short time (being no more than 24h) can't produce remarkable antitumor effect.
Therefore, in the optimized technical scheme, berberine concentration is smaller or equal to 5 μ mol/L in the tumor radio sensitization agent, and further in the optimized technical scheme, berberine concentration is 2 μ mol/L～5 μ mol/L in the tumor radio sensitization agent.
Use the method for above-mentioned tumor radio sensitization medicine, include but not limited to following two kinds of methods:
(1) the berberine injection (as 50mg/kg) of different time (as 30 minutes) intravenous injection various dose before the tumour patient radiotherapy gives radiotherapy then, to improve the radiotherapy curative effect of tumor.
(2) before the tumour patient radiotherapy different time (as 60 minutes) locally injected into tumor various dose contain the berberine hydrosol (as 100mg/kg), give radiotherapy then, to improve the radiotherapy curative effect of tumor.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
Tumor radio sensitization medicine of the present invention can improve the therapeutic effect of tumour radiotherapy, reduces the radiocurable exposure dose of tumour patient, reduces the toxic and side effects that radiotherapy brought, thereby improves patient's life quality.
Description of drawings
Fig. 1 is cell survival rate and berberine consumption and a time chart among the embodiment;
Fig. 2 is the Annexin-V/PI dyeing of berberine cell death inducing among the embodiment and flow cytometry figure as a result;
Fig. 3 finishes Wester blot method after the berberine that adds variable concentrations in the MDA-MB-231 of exponential growth among the embodiment and the MCF-7 cell culture was handled in 24 hours to measure protein expression figure;
Fig. 4 is that MCF-7 and two kinds of cells of MDA-MB-231 among the embodiment (have/no berberine handle) are through the radiating survival curve figure of various dose.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment:
(1) material: human breast cancer cell strain MDA-MB-231 and MCF-7 purchase in U.S. cell collecting center (ATCC), and are preserved and cultivated by inventor place laboratory; The D-MEM culture medium dry powder, calf serum, the pancreatin powder is available from Gibco company; Standard protein molecular weight, SDS-PAGE sample-loading buffer, RIPA protein lysate, TE electrophoretic buffer, 10 * commentaries on classics film liquid, 30%Acry-Bis, Tris-Hcl, Ammonium persulfate. (AP), SDS, tetramethylethylenediamine (TEMED), propidium iodide (PI) are available from the green skies, Jiangsu biotechnology research institute; Dimethyl sulfoxide (DMSO), agarose and Tween-20 are from Shanghai high-tech biological engineering company limited; Caspase-3, antibody such as bcl-2, bax, cyclin D1 are available from U.S. Santa Cruzs biotech company; RNase A and 10 μ g/mL proteinase K are available from U.S. Sigma Aldrich company.
(2) cell culture: MDA-MB-231 and MCF-7 cell culture are in containing 10% calf serum, the D-EME culture medium of glu famine and 1,000,000 U/L penicillins and streptomycin.Cell places 5%CO
2, cultivate in 37 ℃ of incubators, went down to posterity 1 time in every 2-3 days, the trophophase cell of taking the logarithm in the present embodiment is used for experiment.
(3) illuminate condition: cell places under the linear accelerator, and radiation quality is high energy 6 megavolt χ-lines, and irradiation field is 10cm * 10cm, and source-skin distance is 100cm, and close rate is 200cGy/min.Cover the thick equivalent stencil plate of 1.5cm during irradiation on the culture dish face to adjust exposure dose.
(4) mtt assay is observed the growth of breast carcinoma MDA-MB-231 and MCF-7 cell: 24 hour cells (5 * 10 before drug treating
4/ mL) being seeded in 96 orifice plates, each concentration is established 5 parallel holes.The berberine that adds variable concentrations continues to cultivate 24,48 or 72 hours.Adding 20 μ LMTT (5 μ g/mL) in the cell culture fluid continues to cultivate 4 hours.Culture fluid is abandoned in suction, and every hole adds 150 μ L DMSO, and shaking table softly vibrated 10 minutes, adopts microplate reader (A570nm/A630nm light absorption value) to detect optical density at last, adopts cell survival rate=dosing class value/contrast class value * 100% mode to calculate cell survival rate.
The triplicate experiment, experimental result (meansigma methods ± error) will be obtained after three Data Processing in Experiment, draw cell survival rate and berberine consumption and time chart, the result is referring to Fig. 1: berberine obviously suppresses the growth of cell, and manifests the relation of docs-effect and processing time-effect; Berberine suppresses the dosage (IC of 50%MCF-7 cell growth
50) be respectively: 20.1 μ mol/L (24 hours), 10.4 μ mol/L (48 hours) and 5.3 μ mol/L (72 hours); At an other breast cancer cell line MDA-MB-231, berberine IC
50Be respectively 10.3 μ mol/L (48 hours) and 4.7 μ mol/L (72 hours).These experimental result explanation berberines suppress the growth of breast carcinoma MCF-7 and MDA-MB-231 cell significantly, are directly proportional with berberine concentration of treatment and processing time, promptly show as dosage-dependence and processing time dependency.The MDA-MB-231 cell than MCF-7 cell to the little sensitivity of berberine some, but the IC of berberine
50Concentration illustrates that all at about 10 μ mol/L (48 hours) and about 5 μ mol/L (72 hours) it may be the cell line dependent/non-dependent that berberine suppresses the breast cancer cell growth.
(5) variation in Flow Cytometry analysis of cells cycle: breast cancer cell MDA-MB-231 and MCF-7 are inoculated in the 100mm culture bottle, treat that adherent back (treating that cell grows to about 60-70%) adds the variable concentrations berberine respectively and handles to collect after 24 hours and respectively organize cell, adopt 1, centrifugal 5 minutes of 800g centrifugal speed.Supernatant discarded is used 1x PBS buffer washed cell 1 time, and 1, centrifugal 5 minutes of 800g, and collecting cell precipitation.Cell is placed on 70% ethanol of pre-cooling on ice and fixes 4 hours, centrifugal after, with twice of 1x PBS washed cell.After centrifugal, suspension cell contains in the PI solution of 300 μ g/mL RNase A and 10 μ g/mLproteinase K (U.S. Sigma Aldrich company) (10 μ g/mL) in 500 μ L, after 30 minutes, via hole diameter is that the nylon leaching net of 53 μ m filters check and analysis on the FACS-Calibur flow cytometer.Analysis result is referring to table 1: berberine is handled the G0/G1 phase cell cycle arrest of having induced dose dependent, has reduced S phase cell proportion.But G2/M phase ratio is not produced obviously influence.And, in MCF-7 that berberine is handled and two kinds of cells of MDA-MB-231, can observe G0/G1 phase cell cycle arrest.These experimental result explanation berberines can be induced the breast cancer cell Cycle Arrest.Along with the increase of berberine concentration, breast cancer cell S phase ratio descends, and occurs tangible G0/G1 phase cell retardance simultaneously.
Table 1. berberine is to the influence of MDA-MB-231 and MCF-7 cell cycle.
Annotate: existing known cancer cell mainly is that cell cycle arrest is one of the many chemotherapeutics of breast carcinoma and radiocurable important function mechanism owing to cell cycle destroys the property out of control growth that has caused cell.
*P＜0.05 (comparing) with matched group.
(6) Phosphatidylserine turns up, and (the two methods of dying of Annexin V and PI adopt Annexin-V/PI dyeing and flow cytometry method cell can be divided into 4 kinds of attributes, wherein D1 quadrant (Annexin-V to the analysis of cells apoptosis
-/ PI
+) be the non-viable non-apoptotic cell quadrant; D2 quadrant (Annexin-V
+/ PI
+) be non-viable apoptotic cell; D3 quadrant (Annexin-V
-/ PI
-) be injuring normal cell not; D4 quadrant (Annexin-V
+/ PI
-) be viable apoptotic cell.): the cell after cellular control unit and variable concentrations berberine handled is washed 2 times with employing PBS, adds Annexin-V (20 μ g/ml) the 10 μ l of 100 μ l binding buffer and FITC labelling, and room temperature lucifuge 30 minutes.Cell adds PI (50 μ g/ml) 5 μ l again, after lucifuge leaves standstill 5 minutes, adds 400 μ l binding buffer at last, and uses the flow cytometer detection by quantitative immediately.As negative control, analysis result is referring to Fig. 2 with the pipe that do not add AnnexinV-FITC and PI.
From the result of Fig. 2 as can be seen, the cell quantity that is in the IV quadrant at untreated breast carcinoma MDA-MB-231 and MCF-7 cell is considerably less, promptly respectively 5.2% and 3.4%, and the cell that is in I and II quadrant almost be detect less than.Breast carcinoma MDA-MB-231 that berberine was handled in 24 hours and MCF-7 apoptosis and dead cell ratio all increase, at MDA-MB-231 and MCF-7 cell 51.2% and 23.3% (10 μ mol/L) and 86.6% and 66.6% (40 μ mol/L) respectively, promptly the viable apoptotic cell quantity of IV quadrant increases along with the concentration of berberine and is obvious increase.And berberine liquid makes the also showed increased of cell quantity be in I and II quadrant.These experimental results illustrate that heavy dose of berberine can induce the apoptosis of breast cancer cell.
(7) the Western-blot method is analyzed the influence of berberine cell cycle and apoptosis-related protein: collecting cell also went to centrifugal in the Eppendorf of 1.5ml pipe (2500 rev/mins) 5 minutes, abandon supernatant, and add 100 μ l IP lysates, placed 2 hours on ice.4 ℃ centrifugal 5 minutes (2,500 rev/mins) shift supernatant to new Eppendorf pipe, and adopt spectrophotometer test sample protein content.Add albumen sample-loading buffer (5 *) mixing, place on the constant temperature vortex mixer, 100 ℃ 5 minutes, go on the cellulose acetate film behind the protein electrophoresis.Film adopts confining liquid (5% defatted milk powder, 1 * T-BST buffer) sealing 1h.Washing, and add an anti-β-Actin (1: 1000 dilution), Bax (dilution in 1: 1000), Bcl-2 (dilution in 1: 500), Caspase 3 (dilution in 1: 1000), Cyclin B (dilution in 1: 500), the cellulose acetate film room temperature that Cyclin D1 (dilution in 1: 500) handles through confining liquid was fostered 1 hour.The T-BST buffer is washed film 3 times, adds two anti-(dilutions in 1: 1000) and fosters 1 hour.The T-BST buffer is washed film 3 times, adds the ECL luminous agent at last, development, photographic fixing, and after film washing and the drying, scanning analysis.Analysis result is referring to Fig. 3.
As shown in Figure 3, after berberine was handled in 24 hours, the Bax protein expression all manifested the dependent obvious increase of dosage in MDA-MB-231 and two kinds of cells of MCF-7.Equally, the Caspase-3 protein expression level also shows the obvious raising of dose dependent.On the contrary, reducing all appears in two kinds of proteic expression of Bcl-2 and Cyclin B1, handles the back at 5 μ mol/L and just as seen significantly descends, and reduce with the increase of berberine concentration.And berberine does not influence Cyclin D1 protein expression level.
(8) cell clone forming method: the cell that 5 μ mol/L berberines were handled 4 hours is accepted the χ-radiation irradiation of various dose, is inoculated into the 100mm culture dish then.Continuous culture is after three weeks, adopts methanol to fix, and adds Ji's nurse Sa liquid dyeing 30 minutes, and flowing water is cleaned, and observes at last and write down to contain 〉=clone's number of 50 cells.Adopt cloning efficiency (PE)=(matched group clone number/experimental group cell number) * 100% formula to calculate cloning efficiency, adopt surviving fraction=experimental group cloning efficiency/matched group cloning efficiency formula to calculate surviving fraction.Calculate radiation sensitization than SER=matched group D0/ experimental group D0.
Triplicate is tested, and gets the meansigma methods (all error in datas are all below 5%) of three experimental results, adopts the multi-hit model of single target to finish the match survival curve, and the result is referring to Fig. 4.
As shown in Figure 4, compare with the cell that berberine of no use is handled, survival curve appears in the MCF-7 after berberine is handled and two kinds of cells of MDA-MB-231 is not had shoulder, in line.Calculating berberine concentration was handled in 5 μ mol/L24 hours after the multi-hit model curve of single target returned and detects MDA-MB-231 cell and MCF-7 cell radiation sensitivity ratio are respectively 1.4207 and 1.5420.These experimental result explanation berberines increase the radiosensitivity of breast cancer cell.
Annotate, in the present embodiment, all experiments all repeat 3-5 time, average, and the result uses
Expression.Adopt the SAS statistical software that related data is carried out the t check, with P＜0.5 for significant difference is arranged.The clone forms cell survival curve and regression equation employing SigmaPlot 9.0 analyses in the experiment.
Present embodiment proves that berberine is by inhibition tumour cell cycle process and the expression that influences cell cycle progression and cell death related protein, thereby the increase tumor cell is to radiocurable sensitivity.Therefore, the present invention is comprising that at berberine administration and radiotherapy administration simultaneously can improve tumor radio sensitization before the various tumour radiotherapies of breast carcinoma, thereby improves radiocurable therapeutic effect.
Claims (4)
1. the application of berberine in the preparation tumor radio sensitization medicine.
2. a tumor radio sensitization medicine is characterized in that, described tumor radio sensitization medicine active component is a berberine.
3. according to the described tumor radio sensitization medicine of claim 2, it is characterized in that berberine concentration is smaller or equal to 5 μ mol/L in the tumor radio sensitization agent.
4. according to the described tumor radio sensitization medicine of claim 3, it is characterized in that berberine concentration is 2 μ mol/L～5 μ mol/L in the tumor radio sensitization agent.
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Cited By (6)
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| CN106420736A (en) * | 2016-11-14 | 2017-02-22 | 哈尔滨医科大学 | Application of berberine in preparation of drug for treating fibrinolysis system disorder related diseases |
| WO2018224016A1 (en) * | 2017-06-09 | 2018-12-13 | 四川大学华西医院 | Use of berberine or derivative thereof in preparing myocardial perfusion imaging agent |
| CN109394759A (en) * | 2019-01-15 | 2019-03-01 | 王军 | Berberine is as the application prepared in tumor radio sensitization medicine |
| CN114632086A (en) * | 2022-03-07 | 2022-06-17 | 山东大学第二医院 | Application of scutellarin in enhancing radiation sensitivity of iodine-125 particles |
| CN114767756A (en) * | 2022-04-25 | 2022-07-22 | 复旦大学附属华山医院 | Application of coptis chinensis detoxification decoction in preparation of sensitizer for tumor immune checkpoint blockade therapy |
| CN116808031A (en) * | 2023-06-30 | 2023-09-29 | 哈尔滨商业大学 | A Chinese medicinal monomer composition derived from Coptidis rhizoma decoction for treating breast cancer, and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106420736A (en) * | 2016-11-14 | 2017-02-22 | 哈尔滨医科大学 | Application of berberine in preparation of drug for treating fibrinolysis system disorder related diseases |
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| US11141496B2 (en) | 2017-06-09 | 2021-10-12 | West China Hospital, Sichuan University | Use of berberine or derivative thereof in preparing myocardial perfusion imaging agent |
| CN109394759A (en) * | 2019-01-15 | 2019-03-01 | 王军 | Berberine is as the application prepared in tumor radio sensitization medicine |
| CN114632086A (en) * | 2022-03-07 | 2022-06-17 | 山东大学第二医院 | Application of scutellarin in enhancing radiation sensitivity of iodine-125 particles |
| CN114767756A (en) * | 2022-04-25 | 2022-07-22 | 复旦大学附属华山医院 | Application of coptis chinensis detoxification decoction in preparation of sensitizer for tumor immune checkpoint blockade therapy |
| CN114767756B (en) * | 2022-04-25 | 2023-04-07 | 复旦大学附属华山医院 | Application of coptis chinensis detoxification decoction in preparation of sensitizer for tumor immune checkpoint blockade therapy |
| CN116808031A (en) * | 2023-06-30 | 2023-09-29 | 哈尔滨商业大学 | A Chinese medicinal monomer composition derived from Coptidis rhizoma decoction for treating breast cancer, and its application |
| CN116808031B (en) * | 2023-06-30 | 2024-03-15 | 哈尔滨商业大学 | A Chinese medicinal monomer composition derived from Coptidis rhizoma decoction for treating breast cancer, and its application |
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